JXB Advance Access originally published online on April 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 387, pp. 1607-1614,
June 1, 2003
© 2003 Oxford University Press
Changing desiccation tolerance of pea embryo protoplasts during germination
Received 7 October 2002; Accepted 14 March 2003
Department of Biology, The University of South Dakota, 414 E. Clark Street, Vermillion, SD 57069, USA
1 To whom correspondence should be addressed. Fax: +1 605 677 6557. E-mail: kkoster{at}usd.edu
2 Present address: Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
3 Present address: Department of Biology, Northern State University, Aberdeen, SD 57401, USA.
4 This value was originally called the CMC, the critical moisture content, by Reisdorph and Koster (1999). However, the term ‘critical water content’ is often used to describe the water content at which damage first becomes apparent (the ‘threshold water content’ of Reisdorph and Koster). WC50 is a less ambiguous expression for quantifying the 50% damage point.
5 For convenience, sampling times are designated as hours from the onset of imbibition. Germination was completed by 24 h of imbibition, and radicles at 36 h of imbibition were in a post-germinative stage.
6 Arcsin transformation converts 50% to 45%; therefore, 45% survival was used in the transformed data for calculation of critical moisture contents.
7 WC50s were determined on a dry matter basis (g g–1 DW) rather than by equilibration to defined water potentials because the slower drying rates involved in equilibrium drying led to death of the entire population of protoplasts (Xiao and Koster, 2001). Water potentials corresponding to these water contents were extrapolated as described in Table 1.
Abbreviations: ANCOVA, analysis of covariance; DW, dry weight; FDA, fluorescein diacetate; MES, 2-(N-morpholino)ethanesulphonic acid; WC50, water content at which 50% of a population is killed.
Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.
Key words: Desiccation tolerance, embryo, germination, pea, Pisum sativum L., protoplast isolation.
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